RTVP based compositions and methods for the treatment of prostate cancer

ABSTRACT

This invention relates to a gene encoding RTVP that has been shown to be up-regulated by p53 using differential display-PCR and subsequently by co-transfection studies. RTVP-1 mRNA is abundant in normal mouse and human prostatic epithelial cells and primary tumors, but is significantly down regulated in metastatic mouse and human prostate cancer. In prostate cancer cells overexpression of the mouse RTVP-1 gene (mRTVP-1) induced apoptosis that was accompanied by increased caspase 8, 9 and 3 activities. mRTVP-1-stimulated apoptosis was also associated with increased levels of bax, bad and activated BID; reduced levels of bcl-2 and bcl-X L ; and cytosolic cytochrome c accumulation. Adenoviral-vector-mediated mRTVP-1 expression lead to potent growth suppression and antimetastatic activities in an orthotopic mouse model of prostate cancer in vivo. These therapeutic activities were associated with anti-angiogenic effects and importantly a local and systemic immune response. Accordingly, p53 was linked with suppression of metastasis through its induction of mRTVP-1, which can concurrently induce apoptosis, suppress angiogenesis and stimulate an antitumor immune response. Thus, the invention includes compositions and methods, based on RTVP nucleic acid, polypeptides, and antibodies, for use in the treatment, prevention and detection of neoplastic disease and, specifically, metastatic prostatic neoplasia.

REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to U.S. Provisional applicationNo. 60/209,989, filed Jun. 8, 2000.

RIGHTS IN THE INVENTION

[0002] This invention was made, in part, with United States governmentsupport under grant number RO1-50588, awarded by the National CancerInstitute, and also grant number P50-58204, awarded by the NationalCancer Institute, Specialized Program or Research Excellence (SPORE),and the United States Government has certain rights in the invention.

BACKGROUND OF THE INVENTION

[0003] 1. Field of the Invention

[0004] The present invention relates to RTVP genes and associatedsequences, to RTVP proteins, and to methods and tools using thesesequences for the diagnosis, study and treatment of disease. Further,the invention includes RTVP receptor protein and the gene which encodesthis protein. In particular, the invention relates to compositions andmethods based on RTVP for the treatment, prevention and detection ofprostatic neoplasia such as prostate carcinoma and associated metastaticdisease.

[0005] 2. Description of the Background

[0006] The prostate is a walnut size gland that is part of the malereproductive system that makes and stores seminal fluid. It is locatedbelow the bladder, in front of the rectum, and surrounds the upper partof the urethra. To work properly, the prostate needs male hormones(androgens). The main male hormone is testosterone, which is produced bythe testicles. Prostate cancer or PC is characterized by theuncontrolled growth of prostate epithelial cells to form one or moretumors.

[0007] Localized PC is treated very successfully and results in a 100%five-year survival rate. Metastatic PC has a 31% survival rate, about80% of which attacks the bone with a significant portion of theremainder attacking the lungs. PC that metastasizes to the bone is notbone cancer and is treated as later stage or distant PC.

[0008] The American Cancer Society reports that excluding skin cancer,PC is the most common malignancy and the second leading cause of cancerdeath among men in the US. The incidence and mortality of PC increasewith age, 77% of men with new diagnoses of prostate cancer each year areover the age of 65. PC is rare in younger men, with an incidence rate ofless than one case per 100,000 for men under age 40. However, the rateclimbs to 82 per 100,000 for men ages 50-54, 518 for ages 60-64, and1,326 for ages 70-74. African-Americans are twice as likely to developand die from PC than men of other ethnic and racial groups.

[0009] The number of PC cases will increase dramatically during the nextfour decades as the demographics of the Baby Boom generation take effectand the reduction of deaths from cardiovascular and smoking-relateddiseases increase the size of the 60-84 year old population segment. Itis estimated that approximately 570,000 new cases of PC will bediagnosed in 2030 (when there will be close to 50 million men in thispopulation group), and estimated over 2,000,000 men in treatment. Thiscompares to an incidence of 168,665 new cases in 1995.

[0010] Surgery and/or radiotherapy remain the treatments of choice forearly PC. Typically, surgery requires complete removal of the prostate(radical prostatectomy), and quite often removal of surrounding lymphnodes (lymphadenectomy). Radiotherapy, which is also used as adjuvanttherapy, may be either external or interstitial and involves exposure ofthe effected tissue to radioisotopes such as ¹²⁵I. With more advancedforms of PC, endocrine therapy is often the preferred treatment. The aimof this therapy is to deprive prostate cells, and presumably transformedprostate cells as well, of testosterone. This can be accomplished byorchiectomy (castration), or administration of drugs (e.g. leuprolide,goserelin), antiandrogens (e.g. flutamide and bicalutamide), estrogensor synthetic hormones that are agonists of luteinizing hormone-releasinghormone, which directly inhibit testicular and organ synthesis andsuppress luteinizing hormone secretion which in turn leads to reducedtestosterone secretion by the testes. Despite the advances made inachieving a pharmacologic orchiectomy, the survival rates for those withlate stage carcinomas are poor.

[0011] In its more aggressive form, transformed prostatic tissues escapefrom the prostate capsule and metastasize invading locally andthroughout the bloodstream and lymphatic system. Metastasis, defined astumor implants which are discontinuous with the primary tumor, can occurthrough direct seeding, lymphatic spread and hematogenous spread. Allthree routes have been found to occur with PC. Local invasions typicallyinvolve the seminal vesicles, the base of the urinary bladder, and theurethra. Direct seeding occurs when a malignant neoplasm penetrates anatural open field such as the peritoneal, pleural or pericardialcavities. Cells seed along the surfaces of various organs and tissueswithin the cavity or can simply fill the cavity spaces. Hematogenousspread is typical of sarcomas and carcinomas. Hematogenous spread ofprostatic carcinoma occurs primarily to the bones, but can includemassive visceral invasion as well. It has been estimated that a majorityof newly diagnosed prostate cancer patients will have metastases at thetime of initial diagnosis.

[0012] Many studies have shown that there exists a specific metastasissuppressor role for p53, a well-known tumor suppressor protein, in PC(reviewed in⁵). Initial experimental results using an in vivo mousemodel of PC metastasis demonstrated that loss of p53 function can leadto the development of metastases that seed from relatively small numbersof cells within the primary tumor⁶ Subsequent studies demonstrated thatalthough p53 mutations in human primary PC tissues are heterogeneous andrelatively infrequent, they occur at significant levels in metastaticdisease, ranging from 21%-30% mutation frequency in lymph nodemetastasis to higher than 90% mutation frequency in androgen-insensitivedisseminated disease⁷-12. This pattern of mutations suggests that only afew cells harboring p53 mutation in the primary tumor can seedmetastases that clonally expand at distant sites. Consequently, there isa need for improved research tools, diagnostic tools and therapies,useful for the diagnosis, treatment and prevention of PC and metastasisassociated with transformed prostate cells.

SUMMARY OF THE INVENTION

[0013] The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides newdiagnostic, therapeutic and research tools, and methods based on certainproteins related to testes-specific, vespid and pathogenic proteins(RTVP). The invention also relates to compositions and kits comprisingthe RTVP gene, the RTVP polypeptide and active fragments thereof, andantibodies thereto.

[0014] One embodiment of the invention is directed to isolated nucleicacids comprising the sequence of the RTVP gene. The gene may comprisethe entire sequence of RTVP, all or part of the sequence. Further, theinvention includes the RTVP polypeptide encoded therein, and activeportions thereof. Genes may be functionally linked to vectors such asplasmid or viral vectors and be capable of expression in a suitablehost.

[0015] Another embodiment of the invention is directed to recombinantcells containing nucleic acid segments that encodes RTVP polypeptidesand portions thereof. Cells may be eukaryotic or prokaryotic, and mayexpress the protein or act simply as biological containers of the genesequence.

[0016] Another embodiment of the invention is directed to antibodiesreactive against the RTVP protein and to antibodies that may be reactiveto antigenically active portions thereof. Antibodies may be polyclonalor monoclonal, recombinant or synthetic. A further embodiment of theinvention is directed to hybridomas that express antibodies to the RTVPpolypeptide or to antigenic portions thereof.

[0017] Another embodiment of the invention is directed to the receptorof RTVP protein, and to compositions and methods useful for thetreatment of neoplastic diseases such as prostate cancer and relatedmetastasis. A further embodiment is directed to the gene for thereceptor protein as well as to associated promoter and othertranscription or translation controlling sequences. A further embodimentincludes antibodies to the RTVP receptor protein which may also beuseful for the treatment of neoplastic diseases such as prostate cancerand metastasis.

[0018] Another embodiment of the invention is directed to kits for thedetection of prostatic disease. Kits comprise all or characteristicportions of the RTVP gene or the RTVP polypeptide sequence, or toantibodies to either the polypeptide or nucleic acid sequences, and areuseful for detection of disease.

[0019] Another embodiment of the invention is directed to nucleic acidsequences that comprise the anti-sense of the RTVP gene orrepresentative portions thereof. Sequences may be useful in compositionsto for the treatment of prostatic disease by reducing or shutting downRTVP expression in cells.

[0020] Another embodiment of the invention is directed to compositionscomprising RTVP or RTVP receptor polypeptides, or active portionsthereof, as pharmaceutical compositions. Compositions may be useful intherapy, prophylaxis, diagnosis, or as research tools, and may furthercomprise pharmaceutically acceptable carriers for use in the treatmentor prevention of diseases such as prostate cancer and metastaticdisease.

[0021] Another embodiment of the invention is directed to methods forstimulating the immune system such as, for example, cytokines and growthfactors, by administering composition of the invention to patients.Compositions may be administered in a therapeutically safe and effectivedose to humans and other mammals in the form of pills, tablets, powder,liquid or combinations thereof.

[0022] Other embodiments and advantages of the invention are set forthin part in the description which follows, and in part, will be obviousfrom this description, or may be learned from the practice of theinvention.

DESCRIPTION OF THE FIGURES

[0023] FIGS. 1A(a) Nucleic acid sequence of mouse RTVP-1 (SEQ ID NO: 1);and (b) comparison of mouse (SEQ ID NO. 2) with human (SEQ ID NO. 3)RTVP-1 amino acid sequence.

[0024]FIG. 2 Identification of RTVP-1 regulation by p53.

[0025]FIG. 3 In situ hybridization of human prostate cancer.

[0026]FIG. 4 mRTVP-1 induction of apoptosis through mitochondrial deathpathway.

[0027]FIG. 5 RTVP-1 suppression of tumor growth and metastasis.

[0028]FIG. 6 Local and systemic immune response in AdmRTVP-1 treatedtumors.

[0029] FIGS. 7(a) Molecular pathways in prostate cancer, (b) bystandereffect of p53 gene therapy, and (c) p53 regulation of prostate cancer.

[0030]FIG. 8 Wild-type p53 regulation of mRTVP-1 promoter constructs.

[0031]FIG. 9 Induction of mRTVP-1.

[0032]FIG. 10 RTVP-1 expression in mouse and human tissue.

[0033]FIG. 11 Induction of apoptosis by mRTVP-1.

[0034]FIG. 12 Activation of mitochondrial pathways by mRTVP-1.

[0035]FIG. 13 Caspase activation by mRTVP-1.

[0036]FIG. 14 Regulation of apoptosis in prostate cancer.

[0037]FIG. 15 Induction of apoptosis and morphological changes byAdmRTVP-1.

[0038]FIG. 16 Increased survival of mice with orthotopic tumors afterAdmRTVP-1 treatment.

[0039]FIG. 17 Apoptosis in 178-2BMA orthotopic tumors.

[0040] FIGS. 18(a) Immune cell infiltration in 178-2BMA orthotopictumors, and (b) immune response in animals with 178-2BMA orthotopictumors.

[0041]FIG. 19 Cytokine induction by mRTVP-1.

[0042]FIG. 20 Activation of JNK pathway by mRTVP-1.

[0043]FIG. 21 Schematic showing potential mechanisms of mRTVP-1-mediatedsuppression of metastasis.

[0044]FIG. 22 Demethylation of 148-IPA cells induces RTVP-1 expression.

DESCRIPTION OF THE INVENTION

[0045] As embodied and broadly described herein, the present inventionis directed to nucleic acid sequences encoding RTVP or the RTVP receptorprotein, or active portions thereof, to RTVP and RTVP receptorpolypeptides and functional portions thereof, to antibodies to thereto,and to compositions, kits and methods based on RTVP for the treatment,prevention and detection of disease, and specifically prostate disease,in mammals.

[0046] It has been shown that there exists a specific and establishedassociation between loss of p53 function and prostate cancer metastasis.Recent studies demonstrated that specific p53 mutations are clonallyexpanded in metastatic prostate cancer¹¹ and that a pattern of aberrantp53 expression in primary tumors, termed “clustered p53 staining,” hassignificant prognostic value in predicting recurrence following radicalprostatectomy^(12,13) It is generally considered that the nature offunctional alterations which occur in cells containing p53 mutationsspecifically facilitates metastatic seeding, survival, and growth atdistant metastatic sites. These alterations likely result, in part, fromaberrant regulation of genes under the transcriptional control of p53that have previously been shown to mediate apoptosis^(1,14)-17 andanti-angiogenic activities¹⁸-22.

[0047] Surprisingly, it has been discovered that certain proteinsrelated to testes-specific, vespid and pathogenic proteins (RTVP), andspecifically mouse RTVP-1, are up-regulated by p53 in mouse prostatecancer cells. Proteins which are Related to Testes-specific, Vespid andPathogenic proteins include those protein with homology to mammaliantestes-specific proteins (e.g. TPX1), plant pathogenesis-relatedproteins (e.g. PR-protein such as subtype 1, PR-1b), vespid venomallergan proteins (antigen 5), or combinations thereof. Homology meansthat there is a relevant degree of similarity in the amino acid sequencebetween a polypeptide of the invention and one or more of the proteinsmammalian testes-specific proteins, plant pathogenesis proteins andvespid venom allergan antigen 5, or in the respective gene sequences.Relevant homology means that the degree of amino acid sequencesimilarity is about 35% or greater, preferably about 45% or greater,more preferably about 60% or greater, and even more preferably about 75%or greater. Homology can be determined directly by sequencing thepolypeptide of interest and comparing it with the known sequence, orexperimentally by methods well know to those or ordinary skill in theart. Homology can be determined using, for example, blastp queries atdefault settings for amino acid homology determinations, and usingblastn queries at default settings for nucleic acid homologydeterminations. Although the human RTVP gene was previouslyisolated^(23,24), the functional significance of RTVP expression in anybiological context has not previously been reported. Further, human RTVPis significantly homologous (about 50% or greater) to mouse RTVP-1(Table 1). Loss of RTVP-1 expression is associated with mouse and humanprostate cancer metastasis. Importantly, it has also been discoveredthat mRTVP-1 has pro-apoptotic, anti-angiogenic, and immunostimulatoryactivities. This multifaceted role for mRTVP-1 in suppression ofprostate cancer metastasis has also been shown usingadenoviral-vector-mediated mRTVP-1 expression to suppress growth andmetastasis of prostate cancer in vivo. Further, based on the presence ofan extracellular protruding domain of the RTVP protein and the resultsof media-transfer experiments, therapeutic control of disease may alsobe achieved by targeting an RTVP receptor protein.

[0048] Accordingly, the present invention is directed to a novel gene,RTVP, which is under the regulation of p53 in human and mouse prostatecancer cells and potentially other normal and malignant cells. Thesequence was identified using cell lines derived from mouse prostatecancer generated by the mouse prostate reconstitution model system.Briefly, a primary mouse prostate cancer cell line (148-1PA) wasinfected with an adenoviral vector containing wild-type human p53 andwith a control virus without any added exogenous genes. The p53-inducedand control mRNAs were used to generate cDNAs, and a fragment wasisolated using differential display-PCR (DD-PCR) techniques. Thisfragment encoded the novel mouse protein, RTVP-1. Upon sequencing theDD-PCR fragment, it was determined that this mouse cDNA may be relatedto the human mRNA for RTVP-1 protein/human glioma pathogenesis-relatedprotein, as there appeared to be significant homology at the nucleotidelevel.

[0049] Full-length cDNA for RTVP-1 was cloned using a cDNA libraryprepared from 148-1PA cells. Its expression pattern in normal mouseprostate cancer tissues was further analyzed. Expression for thisprotein at the mRNA level appeared to be relatively low in most tissuesexamined including prostate, yet mRNA levels were exceedingly high forcolon, spleen and lung. Further analysis of primary and metastatic mouseprostate cancer cell lines by Northern blotting indicated that RTVP waspresent at low levels in primary tumor-derived cell lines, but appearedto be down regulated in metastasis-derived cell lines. This indicatedthe possibility that loss of expression of this gene may be selected forduring metastatic progression of prostate cancer.

[0050] To determine the potential functional significance of RTVP,adenoviral vectors containing the mouse RTVP cDNA were constructed andthis adenoviral vector together with a control vector for apoptoticactivity were tested in the p53 null human colon cancer cell line,H1299. The data clearly indicated that, at MOI of 100, more than 2-foldincrease in annexin V positive cells was seen in RTVP infected cellsrelative to the same MOI of a control virus that expressed only thebeta-galactosidase gene. Additional studies were also consistent withRTVP stimulated apoptosis. These data indicated that a novelp53-regulated gene that is involved in apoptosis in prostate cancer andpotentially other malignancies had been identified. That sequence, andthe peptide it encodes, provide potential diagnostic/prognostic toolsand/or therapeutic targets in clinical malignancies and growthdisorders.

[0051] Accordingly, one embodiment of the invention is directed tonucleic acids that comprises all or one or more portions of the sequenceof RTVP, to peptides derived from these sequences, and to sequencescomplementary thereto. The nucleic acid sequence that encodes mouseRTVP-1 protein is shown in FIG. 1a (SEQ ID NO: 1). The amino acidsequence of the mouse RTVP-1 protein (SEQ ID NO. 2) is shown in FIG. 1b.Nucleic acids of the invention may be single-stranded or double-strandedand composed of DNA, RNA or PNA, or another appropriate nucleic acid,polypeptide or functionally similar backbone structure. Single strandednucleic acids may be in the form of a sense strand or an antisensestrand. Further, although the human RTVP gene sequence is know (GenBankDNA database accession number X91911), RTVP genes of the invention maybe derived from other mammals such as, for example, mice, rats or anyrodent, cattle, sheep, goats, pigs, horses, canines, felines, and mostany other non-human mammal.

[0052] Nucleic acids according to the invention include isolated (e.g.purified) and recombinant nucleic acid sequences comprising SEQ ID NO:1, or a portion of SEQ ID NO: 1 encoding all or one or more activefragments of the RTVP polypeptide. Nucleic acids containing conservedregions of sequences and nucleic acids encoding open reading frames andconserved domains within open reading frames are typically sufficient torepresent or contain identifiable portions of RTVP such as functionaland antigenic portions. Nucleic acids may comprise additional sequencessuch as RTVP-specific promoters, activator and repressor sites, andenhancers for modulation of expression of sense or antisense messages,recombination sequences for gene targeting, selectable markers fortransfections, or replication origins for passage in a host such asbacteria, virus, eukaryotic cells or yeast. A further embodiment of theinvention includes RTVP-specific promoters which modulationtranscription (e.g. by differential methylation of promoter sequences)of RTVP in normal, pre-malignant and malignant cell. These promoters canbe functionally coupled to anti-neoplastic genes to treat or preventcell proliferative disorders such as, for example, tumors, prostatecancer, and metastatic disease. Nucleic acids may be packaged in a viralvector such as, for example, a retroviral, a vaccinia or an adenoviralvector. In one embodiment, the sequence may be part of a homologousrecombination vector designed to recombine with another sequence. Theinvention further includes vectors comprising the nucleic acid sequencesof the invention, polypeptides expressed by these vectors, andrecombinant cells comprising these vectors.

[0053] Another embodiment of the invention is directed to all or aneffective fragment of a RTVP polypeptide comprising all or part of theamino acid sequence encoded from the nucleic acid sequence of FIG. 1a(see FIG. 1b; SEQ ID NO. 2), such as, for example, exonic fragments,fragments encoded in open reading frames of RTVP genes, conserveddomains of the polypeptide or the nucleic acid, and p53 binding sites.Specifically, the invention includes isolated polypeptides comprisingall or active fragments of the polypeptide encoded in an RTVP gene, andmonoclonal and other antibodies to these peptides.

[0054] Nucleic acids and polypeptides or proteins (RTVP and receptorproteins) according to the invention may be used as a diagnostic ortherapeutic tool in the detection, treatment or prevention of diseases,such as neoplastic disorders (e.g. malignant tumors, metastatic disease)and cell or tissue growth disorders. For example, one embodiment of theinvention is directed to diagnostic aids or kits for the detection ofneoplasia in a patient. Detection kits may comprise RTVP nucleic acidsequences or RTVP polypeptides whose presence or absence in the samplewould be indicative of the presence of a disease such as, for example,prostate cancer or a prostate metastasis. Samples which can be analyzedinclude samples of biological fluids (e.g. blood, plasma, interstitialfluid, urine, cerebrospinal fluid) and samples of biological tissue(e.g. surgical biopsy).

[0055] Another embodiment of the invention is directed to antibodiesspecifically reactive against polypeptides and proteins and fragmentsthereof of the invention. Antibodies may be polyclonal or monoclonal ofany isotype (e.g. IgA, IgD, IgE, IgG1, IgG2a, IgG2b, or IgM), orantibody fragments (e.g. Fab, Fv), humanized antibodies, or recombinantor synthetic antibodies (e.g. variable region fragments). Further, theinvention comprises hybridomas that express antibodies specificallyreactive against polypeptides of the invention.

[0056] Another embodiment of the invention is directed to a method fortreating a neoplastic or growth disorder comprising administering apharmaceutically effective amount of a composition comprising an RTVPpolypeptide, polypeptide fragment or ligand of a polypeptide. Suchcompositions may be anti-angiogenic, be used to modulate (e.g. increaseor decrease) cytokine or other immune system regulatory proteins ortheir activity, induce apoptosis, and/or stimulate a cell or humoralresponse. For example, in cell growth disorders such as prostate cancerand other neoplasias, cytokine expression may be improperly turned off(e.g. methylated) in malignant or pre-malignant cells. As such, thesegene products may also be useful as a diagnostic for malignancy.Alternatively, compositions of the invention may be useful in, forexample, auto-immune disease to turn on genes whose activity may beimproperly reduced or turned off (e.g. by methylation). Further,polypeptides of the invention may stimulate apoptosis and are useful astherapeutics to treat and prevent neoplasia such as, for example,tumors, metastasis and any uncontrolled cell growth. A furtherembodiment of the invention comprises the RTVP promoters which aredifferentially modulated (e.g. by methylation) and regulate expression.

[0057] The polypeptide or polypeptide fragment, or a ligand to thepolypeptide may be administered by injection, pulmonary absorption,topical application and delayed release. The composition may furthercomprise a pharmaceutically acceptable carrier such as water, alcohols,salts, oils, glycerols, fatty acids, starchs, saccharides,polysaccharides or combinations thereof. More than one carrier may beused together to create a pharmaceutical with desirable properties. Afurther embodiment of the invention comprises vaccines for the treatmentand/or prevention of neoplastic disease. Vaccines may compriseantibodies reactive against polypeptides and/or polypeptide fragments ofthe invention, of the polypeptides themselves. Vaccines comprisetherapeutically effective doses of the therapeutic agent, which may bethe polypeptide or polypeptide fragment, or an antibody or collection ofantibodies which bind or are otherwise reactive thereto.

[0058] Another embodiment of the invention is directed to nucleic acidsderived or based on the sequence of RTVP such as, for example, thesequence of SEQ ID NO: 1, useful in treatment or diagnosis and indiagnostic kits. Treatment may involve using the sequences, or effectiveparts thereof, in gene therapy, including gene ablation, gene expressionand gene suppression, such as antisense suppression. Diagnosis mayinvolve genotypic analysis of samples to determine the existence andexpression levels of the genes. Nucleic acids of the present inventionmay be used in various treatment and research modalities, including genereplacement, gene targeting, antisense inhibition, antisense blocking,genetic ablation and gene silencing. Gene replacement involves replacinga copy of a defective gene with another copy by homologousrecombination. Gene targeting involves the disruption of a cellular copyof a gene by homologous recombination. Gene targeting refers to aprocess of introducing a nucleic acid construct into a cell tospecifically recombine with a target gene in the cell. The nucleic acidconstruct inactivates the gene after targeting. Inactivation may be byintroduction of termination codons into a coding region or introductionof a repression site into a regulatory sequence. Antisense inhibitionexploits the specificity of hybridization reactions between twocomplementary nucleic acid chains to suppress gene expression. If acloned gene is engineered so that only the opposite DNA strand istranscribed, the resultant RNA may hybridize to the sense RNA andinhibit gene expression. Antisense blocking refers to the incorporationinto a cell of expression sequences which direct the synthesis ofantisense RNA to block expression of a target gene. Antisense RNAhybridizes to the mRNA of the target gene to inhibit expression. Geneticablation (gene knockout) refers to one process of silencing a gene in acell. Genetic ablation (gene knockout) may be performed after a cell isselected for use or by selecting a cell already comprising a genotypewith the proper genetic ablation. Ablation of the gene encoding RTVP,for example by pre-transcriptional inhibition (such as homologousrecombination with endogenous recessive oncogenes) or posttranscriptional inhibition (such as the expression of antisenseoncogenes to suppress translation) may be useful. Gene silencing isperformed by transfecting cells with nucleic acids which cause geneticablation or by antisense suppression. The silencing process may includeprocesses such as gene targeting or antisense blocking.

[0059] Another embodiment of the invention is directed to methods tomodulate a cytokine activity with effective amounts of RTVP protein oractive portions thereof. Cytokines whose activity may be up regulated(e.g. through demethylation) include the type 1 or TH1 cytokines suchas, for example, INF-alpha, beta and gamma, TNF-gamma, IL-2, IL-6,IL-12, and the death domain proteins such as Fas, and their ligands(i.e. receptors) such as Fas-ligand and Trail. Cytokines whose activitymay also be modulated by RTVP include the type 2 or TH2 cytokines suchas, for example, IL-1β, IL-4, IL-10. Increased or decreased expressionor function of cytokines is an important aspect of RTVP therapy and hasimplications for both cancer and non-cancer therapy. One of the moreimportant cytokines induced is IFN-gamma, wherein RTVP-1 is believed tobe inducing a methylation change in the IFN-gamma promoter, which isnormally completely methylated and therefore silent in normal cells.Differential methylation of these and RTVP promoter sequences can beused diagnostically to detect as well as therapeutically to treatneoplastic disease. Alternatively, in specific cancer cells includingprostate cancer, global perturbations in methylation can silence tumorsuppressor genes such as, for example, p6, and also lead todemethylation of genes that (i) produce a more malignant cell and istherefore selected during progression (e.g. such as caveolin-1), or (ii)make the cell susceptible to apoptosis when an appropriate stimulus isprovided. In prostate cancer, the IFN-gamma gene promoter may bedemethylated and therefore susceptible to induction. RTVP-1, which isexpressed in normal prostatic epithelial cells, then becomes lethal tothe cell by activating IFN-gamma which leads to direct cell killing orindirect cell killing via an immune response. Upon isolation of the RTVPreceptor, ligands could be identified that could activate the RTVP-1pathway, turn on IFN-gamma or another cytokine, which may be cancer andimmune cell specific and thereby specifically target cancer cells.

[0060] Another embodiment of the invention is directed to receptorprotein for RTVP proteins such as an RTVP-1 receptor, which is importantfor stimulating downstream activities of RTVP-1 Downstream activitiesincludes apoptosis and stimulation of cytokine expression and apoptosisthrough stimulation of an immune response, which offers anextra-cellular therapeutic approach.

[0061] The following examples illustrate embodiments of the invention,but should not be view as limiting the scope of the invention.

EXAMPLES Example 1 Identification of mRTVP-1 as a p53 Target Gene

[0062] Over the last decade, numerous studies have established p53mutations as being paramount to the development and progression ofvarious malignancies. The p53 tumor suppressor protein has beenassociated with various antitumor activities that include growthsuppression, apoptosis, and anti-angiogenic activities. The majority ofhuman tumor mutations decrease the sequence-specific DNA binding andtranscriptional activity of the p53 protein²⁶. Today the large andexpanding list of genes under the transcriptional control of p53 havebeen reported and it has been estimated that 200-300 genes are under thetranscriptional regulation of p53²⁷ To date, the number of reportedp53-regulated genes is approximately 80²⁸.

[0063] To identify prostate cancer-related genes under thetranscriptional regulation of p53, a model system was established usingadenoviral vector delivery of wild-type p53 compared to β-gal or anempty cassette together with differential display-PCR. Using thisapproach, numerous sequences were isolated that were known to be underp53 control including cyclin G, epoxide hydrolase, and MDM2. Inaddition, genes were isolated that had not been previously associatedwith p53 regulation. One of the sequences identified encoded the mousehomologue for RTVP-1^(23,24). Briefly, mouse prostate cancer cell lineswere grown in DMEM with 10% fetal bovine serum (FBS). Human prostatecancer cell lines Tsu-Pr1 and LNCaP were grown in RPMI 1640 with 10% FBSand PC3 in Kaighn's modified am's F12 with 10% FBS. For adenoviralinfections the vector was diluted to the appropriate multiplicity ofinfection (MOI) in serum free medium with 0.1% bovine serum albumin andleft in contact with the cells for 4 h followed by fresh media.Differential display-PCR was as previously described 34 using RNAisolated from AdCMVp53 or Ad gal infected 148-IPA cells. Differentiallyexpressed PCR band was subcloned, sequenced and used to screen mouse andhuman cDNA and mouse genomic libraries. Mouse mRTVP-1 cDNA was insertedinto the plasmid pcDNA3.1 (Invitrogen, Carlsbad, Calif.) fortransfection studies or used to prepare an adenovirus (AdmRTVP-1) aspreviously described^(35,36). Genomic DNA containing mRTVP-1 promoterfragments were subcloned into the luciferase reporter vector pGL3-luc(Promega, Madison, Wis.) and equimolar concentrations transfected intoTsu-PR1 cells with Lipofectamine along with mutant or wild type p53expressing plasmids³⁷. Luciferase activity was standardized to acotransfected galactosidase plasmid as previously described³⁸. Freshlyisolated rat aortic ring slices were infected with adenoviral vector inserum free endothelial medium (EGM-2, BioWhittaker, Walkersville, Md.)for 3 h then cultured in Matrigel for 48 h to allow for endothelial cellsprouting as described 39. Protein lysates were prepared with TNES lysisbuffer [50 mM Tris (pH 7.5); 2 mM EDTA, 100 mM NaCl, 1% NP40, 20 g/mlaprotinin, 20 g/ml leupeptin and 1 mM PMSF], separated on 10-12%polyacrylamide-SDS gels and electrophoretically transferred tonitrocellulose membrane for western blotting. Antibodies included Badand phospho-Bad (New England Biolabs, Beverly, Mass.); BID (R&D Systems,Minneapolis, Minn.); Bax, Bcl-2, Bcl-X_(L), caspase 6 and cytochrome c(BD-Pharmingen/Transduction Labs, San Diego, Calif.); and caspases 3, 7,8, 9, and 10 (Oncogene Research Products, Boston, Mass.). β-actinmonoclonal antibody (Sigma, St Louis, Mo.) was used as a loadingcontrol. Rabbit polyclonal antibody to peptides 59-71 of the mRTVP-1protein was affinity purified. RTVP-1 cDNA was originally cloned fromhuman glioma tissue and was subsequently reported to be expressed indifferentiated macrophages²⁵. Shown in FIG. 2 is the identification ofRTVP-1 regulation by p53. Kinetic analysis of mRTVP-1 mRNA expressionfollowing infection of the p53 null mouse prostate cancer cell line148-IPA⁶ with no adenoviral vector, control adenoviral vector (Ad5CMV),or p53 expressing Ad5CMVp53⁴² (FIG. 2a). Induction of mRTVP-1 byγ-irradiation in p53 wild type prostate cancer cell line RM9⁴³ (FIG.2b). Induction of RTVP-1 by Ad5CMVp53 in the human prostate cancer cellline Tsu-Pr1 relative to uninfected, control Adβgal, or AdmRTVP-1 (FIG.2c). Comparison of deduced protein sequence for mouse and human RTVP-1(FIG. 2d). Identical amino acids are enclosed in dark boxes, conservedamino acid substitutions are lightly shaded, and gaps in the alignmentby a -. A potential N-glycosylation site is indicated by a *. Theputative signal sequence precedes a cleavage site indicated by thesymbol ^ . Two conserved regions known as extracellular proteinsignature motifs 1 and 2 (sig1 and sig2) and a transmembrane domain (TM)are indicated. Schematic of the genomic sequence with potential p53binding sites and fragments used for luciferase constructs (FIG. 2e).The mRTVP-1 genomic sequence was from clone 163K10 from the mouseRPCI-21 PAC library 44. DNA sequence analysis identified multiple p53consensus binding sites (RRRCWWGYYY n RRRCWWGYYY, SEQ ID NO. 4; whereR=purine, Y=pyrimidine, W=A or T and n=1-22 nucleotides) as well asrecognition sites for IFNβ, NFκB, GM-CSF and AP-1 (not shown). Theluciferase activity was determined 24 h after transfection ofmRTVP-1-luciferase expression vectors into Tsu-Pr1 cells along with aβ-gal plasmid for standardization and a plasmid expressing mutant p53(shaded boxes), wild type p53 (solid boxes), or control plasmid (openboxes). A p21 promoter luciferase plasmid was used as a positive controlfor p53 activation.

[0064] Using the differential display-PCR fragment as a probe, it wasdetermined that mRTVP-1 mRNA (˜1.1 kb transcript) was significantlyinduced at 24 and 48 hours after AdCMVp53 infection in the p53 nullmouse prostate cancer cell line 148-1 PA and by γ-irradiation in mouseprostate cancer RM-9 cells that contain wild-type p53. Additionalstudies showed that RTVP-1 (˜0.8 and 1.3 kb transcripts) expression wasalso induced following AdCMVp53 infection in the human prostate cancercell line Tsu-Pr1. Differential display-PCR fragment was used to screena cDNA library generated from 148-1 LMD mouse prostate cancer cells andisolated multiple cDNAs containing the complete ORF of mRTVP-1 thatencodes 255 amino acids with 68% identity to the human RTVP-1 protein.Notably, mRTVP-1 contains two short in-frame deletions of two aminoacids (PH) at positions 86, 87 and a nine-amino acid deletion (KVSGFDALSSEQ ID NO. 5) from amino acid 158 through 166 relative to human RTVP-1.Both mouse and human proteins contain a putative N-linked glycosylationsite and a hydrophobic region near the carboxy terminus. Interestingly,both proteins also contain putative N-terminal signal peptides andextracellular protein signature motifs, which suggests that bothproteins are potentially located on the surface of the cell membrane oreven secreted. The mRTVP-1 cDNA was also used to isolate genomic mRTVP-1and sequenced 2.5 kb of promoter sequences as well as exon 1, intron 1and exon 2 of the mRTVP-1 gene. Multiple putative p53 binding sites weredocumented in the mRTVP-1 promoter and intron 1, with at least 13 inintron 1. In co-transfection studies using mRTVP-1-luciferase expressionvectors, specific induction of luciferase activity by exon 1-intron 1sequences following co-transfection mRTVP-1-Int1-luc with wild-type p53in Tsu-Pr1 cells was demonstrated. When 1.4 or 2.4 kb of promotersequences was included upstream of exon 1-intron 1, a significantincrease in p53 inducibility, but also basal promoter activity wasobserved. These results suggest that one or more putative p53 bindingsites within intron 1 are mediating p53 regulation of mRTVP-1.

Example 2 RTVP-1 mRNA Levels are Down-regulated in Prostate CancerMetastases

[0065] Having demonstrated that mRTVP-1 is induced by p53; it was nextdetermined the mRNA expression profile of RTVP-1 in mouse and humanprostate cancer tissue specimens by in situ hybridization (FIG. 3).Briefly, normal mouse prostate or prostate cancer developed in the mouseprostate reconstitution model with strain 129/SV wild type, heterozygousor homozygous for p53 knockout⁶ were used for in situ hybridization.Human primary prostate cancers and lymph node metastatic deposits aswell as histologically normal prostate were obtained at radicalprostatectomy. The specimens were frozen in liquid nitrogen immediatelyafter surgical removal and 6- μm sections cut with a cryostat. Sectionswere air-dried and fixed in a solution containing 3 parts 4%paraformaldehyde, 4 parts ethanol and 3 parts glacial acetic acid for 20min. Anti-sense cRNAs of mRTVP-1 (400 bases) and hRTVP-1 (256 bases) orcorresponding sense RNA probes were made by run-off transcription of thevector pCR2.0 (Invitrogen) with SP6 or T7 polymerase, respectively usingthe DIG-RNA labeling kit (Roche Diagnostics Corp. Indianapolis Ind.).The sections were prehybridized in 50% formamide, 5× SSC, 5× Denhardt'ssolution, 250 g/ml yeast t-RNA, 4 mM EDTA and 1 mg/ml salmon sperm DNAat 37C° for 60 min and hybridized in the prehybridization buffer(without the salmon sperm DNA) containing 2.5 to 10 ng/l DIG-labeledcRNA probes at 48 C° overnight. Sections were then rinsed in 4× SSC(2×10 min), 2× SSC (10 min) and 1× SSC (10 min) and 0.1× SSC (30 min at48° C.). The DIG-labeled RNA was detected with mouse anti-DIG IgGfollowed with a DIG conjugated antibody to the mouse IgG Fab fragmentand finally anti-DIG IgG conjugated with fluorescein (Roche). Sectionsfrom normal prostate and cancer as well as metastatic cancer depositswere always processed in parallel under the same conditions and usingthe same batches of probes and reagents. Sections were evaluated under afluorescence microscope and the RTVP-1 mRNA levels were scored accordingto the relative fluorescence intensities (see Table 2) as −: No signaldetectable, +: weak; ++ moderate, and +++: strong. Statistical analysisusing Mann-Whitney U test was performed to determine the significance ofthe differences in the fluorescence scores in different tissues.

[0066] Shown in FIG. 3 is RTVP-1 mRNA expression in mouse (m) and human(h) prostatic tissues as demonstrated by in situ hybridization withfluorescent riboprobes. RTVP-1 mRNA is expressed in the basal andglandular epithelial cells of both normal mouse and human prostates(mNP-AS or hNP-AS respectively). Cancer cells expressed a moderate levelof RTVP-1 mRNA in both mouse (mPCa-AS) and human (hPCa-AS) primaryprostate tumors. In contrast, much lower levels of RTVP-1 mRNA wereshown in the metastatic deposits of both mouse (mPCa-Met AS) and human(hPCa-Met AS) lymph nodes. Sections from both mouse and human prostatecancers that were incubated with the sense riboprobes gave rise tominimal signal (mPCa-S and hPCa-S). Original magnification: 200×.

[0067] Semi-quantitative in situ hybridization analysis of a panel ofmouse (not shown) and human prostate cancer tissue specimens revealedabundant RTVP-1 mRNA levels in normal human prostatic basal andsecretory epithelial cells and in primary tumor cells (Table 2).

[0068] However, RTVP-1 mRNA levels were significantly reduced inmetastases relative to normal prostatic epithelium or primary prostatecancer cells. Immunohistochemical analysis for p53 protein using thecriteria of clustered staining¹⁰ revealed a general trend towardassociation of reduced RTVP-1 mRNA and aberrant p53 proteinaccumulation, yet the data were more consistent with independentinactivation of RTVP-1 (Table 2). Extensive sequencing analysis ofRTVP-1 cDNAs isolated from prostate cancer cell lines and tissues didnot reveal RTVP-1 gene mutations suggesting other mechanisms of geneinactivation (data not shown).

[0069] These data show that endogenous RTVP-1 mRNA expression isspecifically reduced in metastatic mouse and human prostate cancer.Interestingly, RTVP-1 mRNA levels were abundant in normal prostateepithelial cells and in primary prostate cancer cells suggesting thatthe RTVP-1 gene is active in the absence of high levels of p53 protein.Therefore, it appears that p53-independent pathways can regulate RTVP-1expression under both normal and abnormal conditions. Immunostaininganalysis of p53 protein showed a trend towards an association betweenaberrant accumulation and reduced RTVP-1 mRNA levels yet the data weremore consistent with independent inactivation of RTVP-1 gene expression.Initial sequencing analysis of cDNAs isolated from malignant prostatecancer cells failed to reveal any structural mutations in the RTVP-1coding region.

Example 3 Pro-apoptotic Activities of mRTVP-1 are Mediated through theMitochondrial Pathway

[0070] To analyze the biological activities of mRTVP-1, a series ofhuman prostate cancer cell lines (Tsu-Pr1, LNCaP, and PC3) and the humanlung cancer cell line, H1299, were infected with AdmRTVP-1 or Adβgal.Western blotting analysis demonstrated high levels of intracellularmRTVP-1 protein by 48 hours after infection.

[0071] As shown in FIG. 4, mRTVP-1 induces apoptosis throughmitochondrial death pathway. Apoptosis was determined by annexin Vbinding and flow cytometry on cells infected with Adβgal (open boxes) orAdmRTVP-1 (closed boxes) at an MOI of 100 as described 38 (FIG. 4a).Protein levels of mRTVP-1 in Tsu-Pr1 cells were determined by westernblotting. Western blotting for bcl family member proteins and cytosoliccytochrome c in LNCaP lysates following transfection with controlplasmid pcDNA or pmRTVP-1 (FIG. 4b). Relative caspase activity wasdetermined by densitometric analysis of western blots of the cleavageproduct for each specific caspase relative to β-actin as a loadingcontrol in lysates from LNCaP cells following infection with Adβgal(open boxes) or AdmRTVP-1 (closed boxes) (FIG. 4c).

[0072] Thus, AdmRTVP-1 induced apoptosis as evidenced by significantincreases in annexin V positivity, DNA fragmentation (not shown) andDAPI staining (not shown). To further probe the mechanisms of apoptosisinduction by mRTVP-1, the expression of bcl-2 family proteins in LNCaPcells was evaluated following mRTVP-1 transfection. These resultsdemonstrated that mRTVP-1 expression led to increased levels ofpro-apoptotic bax, bad and activated BID, but down regulation ofanti-apoptotic bcl-2 and bcl-X_(L). Increased levels of cytosoliccytochrome c were also demonstrated following mRTVP-1 transfection,indicating activation of the mitochondrial apoptosis pathway. Theactivation of specific caspases was also analyzed using western blottingwith antibodies specific for the activated forms of the initiationcaspases 8, 9, and 10 and effector caspases 3, 6 and 7. The resultsindicated that AdmRTVP-1 infection was capable of inducing activation ofcaspases 8, 9 and 3 relative to control Adβgal infection in LNCaP cells.Following mRTVP-1 transfection, apoptosis was also induced withfrequencies and characteristics similar to that observed followinginfection with AdmRTVP-1 (not shown).

Example 4 Adenoviral Vector-mediated mRTVP-1 Expression SuppressesProstate Cancer Growth and Metastasis

[0073] To determine possible therapeutic activities induced byoverexpression of mRTVP-1 in vivo, an orthotopic mouse model ofmetastatic prostate cancer was used. Briefly, orthotopic tumors wereinitiated by injecting 5000 178-2BMA cells into the dorsolateralprostate of 129/SV mice. Seven days later when the tumors achieved anaverage wet weight of 25 mg they were injected with adenoviral vector ina volume of less than 25 μl. Six to ten animals were injected with eachdose. At the indicated times, tumor tissue was weighed then frozen inliquid nitrogen or fixed in 10% formalin and processed for paraffinembedding. Apoptosis was determined by TUNEL labeling as previouslydescribed⁴⁰. Mean vessel density and tumor cell infiltrate quantitationwas essentially as previously described⁴¹ using monoclonal Rat-antimouse antibodies for CD8, CD4, F4/80, TNF-(BD-Pharmingen), as well aspolyclonal antibodies to NOS2 (BD-Pharmingen) and Factor VIII-relatedantigen (Dako, Carpinteria, Calif.). Spontaneous metastases to the lungwere microscopically counted after overnight fixation in Bouin'ssolution⁴¹. NK activity was determined by lysis of YAK cells bysplenocyte derived cells as previously described⁴¹. Serum IL-12 wasdetermined by immunoassay (Biosource, Camarillo, Calif.). All mice weremaintained in facilities accredited by the American Association forAccreditation of Laboratory Animal Care and all experiments conducted inaccordance with the principles and procedures outlined in the NationalInstitutes of Health's Guide for the Care and Use of Laboratory Animals.

[0074] As indicated, seven days following orthotopic inoculation of178-2BMA cells, tumors were injected with either 1×10⁸ or 5×10⁸ PFU ofAdmRTVP-1 or Adβgal. At 7 and 14 days post vector treatment, tumors wererecovered from animals and both primary tumors and their metastases wereanalyzed extensively. As shown in FIG. 5, mRTVP-1 suppresses tumorgrowth and metastasis. Wet weight of orthotopic 178-2BMA tumors treatedwith Adβgal at 1×10⁸ PFU (open box) or 5×10⁸ PFU (hatched box) or withAdmRTVP-1 at 1×10⁸ PFU (shaded box) or 5×10⁸ PFU (closed box) on day 7(left panel) or day 14 (right panel) after tumor cell inoculation. *P≦0.05; ** P≦0.01 (FIG. 5a). Apoptotic index as determined by TUNELlabeling (symbols as in a) (FIG. 5b). The tumor microvessel density(symbols as in a) as measured by factor VIII staining was decreased byhigh dose AdmRTVP-1 (FIG. 5c). Rat aortic ring slices were incubatedwith the indicated dose of adenoviral vector then placed in Matrigel and48 h later photographed (FIG. 5d). Spontaneous lung metastases fromadenoviral vector treated orthotopic tumors in animals was determined onday 21 (symbols as in a) (FIG. 5e).

[0075] AdmRTVP-1 significantly suppressed the growth of primary tumorscompared to control Adβgal vector injections at both vector doses whenevaluated at both time points (P≦0.01). Additional analysis indicatedthat increased apoptotic levels were likely responsible for the growthsuppressive effects of AdmRTVP-1 as significant increases in TUNELlabeling were documented in tumors treated with 5×10⁸ PFU of AdmRTVP-1at both day 7 (P=0.028) and day 14 (P=0.015). Further, a significantreduction (P=0.004) in the density of Factor VI-positive tumorassociated endothelium was also associated with the higher dose ofAdmRTVP-1 when day 14 tumors were analyzed. In an independent in vitroangiogenesis assay, the rat aortic ring sprouting assay, treatment withAdmRTVP-1 inhibited endothelial cell sprouting compared to Adβgal. Toevaluate potential antimetastatic effects of mRTVP-1 in the 178-2 BMAorthotopic model, the extent of lung metastases in AdmRTVP-1-treated andcontrol Adβgal-treated animals was analyzed on day 14 after tumorinitiation. Both doses of AdmRTVP-1 significantly suppressed spontaneouslung metastases compared to Adβgal infection (P≦0.002), indicating thatmRTVP-1 associated activities lead to suppression of growth andspontaneous metastatic activities of orthotopically grown mouse prostatecancer. These results suggest that mRTVP-1 mediated pro-apoptotic andanti-angiogenic activities likely played a role in suppression of tumorgrowth and metastasis.

Example 5 Expression of mRTVP-1 Induces a Local and Systemic Anti-tumorImmune Response

[0076] To further explore the underlying mechanisms responsible formRTVP-1 mediated antimetastatic activities, local and systemic immuneresponses was analyzed in animals with AdmRTVP-1 treated tumors comparedto control Adβgal infected tumors. Local and systemic immune response inAdmRTVP-1 treated tumors are shown in FIG. 6. Immunohistochemicaldetection of F4/80, TNF-α, NOS2 (iNOS), CD4 and CD8 positive cells werequantitated by image analysis and expressed as positive cells per mm²(symbols as in FIG. 5) (FIG. 6a). NK activity two days after vectorinoculation. (▪=AdmRTVP-1 treated animals,

=Adβgal treated animals) (FIG. 6b). Serum IL-12 levels from animalssacrificed on day two or three after vector inoculation (FIG. 6c).

[0077] The results indicated significant increases in the density oftumor-associated F4/80positive macrophages (P=0.03 for 1×10⁸ PFU andP-0.01 for 5×10⁸ PFU) and CD8⁺ T cells (P=0.004 at both doses) inAdmRTVP-1 infected tumors relative to control Adβgal infected tumors atboth day 7 following vector treatment. Significant infiltrates of tumorassociated macrophages and CD8+ T cells persisted for both doses at 14days post treatment (P=0.017 and P=0.02 for macrophages and P=0.018 andP=0.0005 for CD8+T cells for 1×10⁸ PFU and 5×10⁸ PFU respectively)indicating sustained immunostimulatory activities. Analysis ofactivation markers associated with tumor associated F4/80-positivemacrophage indicated that AdmRTVP-1 specifically increased NOS-2 andTNF-α activities in this cell type. To determine whether mRTVP-1overexpression in the primary tumor elicited a systemic immune responsesplenocytes were isolated from mice bearing AdmRTVP-1 and control Adβgalinfected tumors and used for analysis of NK activities. Significantlyincreased NK activities were demonstrated two days post vector injectionin the mice with AdmRTVP-1 treated tumors compared to mice with Ad galtreated tumors (P=0.01, unpaired t test at E:T of 100:1 and 0.004 at50:1) In addition, a significant increase in serum levels of IL-12(P=0.001) was seen in animals with AdmRTVP-1 treated tumors relative toAdβgal two to three days following vector injection.

[0078] Overall, these results specify that mRTVP-1 as a p53-induced geneand is involved in apoptosis-mediated tumor suppressor activities.Although there are numerous studies that have documented the tumorsuppressing activities of p53 in various systems and clear evidence hasbeen found for a role for p53 as a tumor suppressor gene in humancancer, the reported antimetastatic activities of p53 have thus farremained somewhat ambiguous. Certainly, the induction of apoptosis andsuppression of angiogenesis could inhibit metastatic spread, yet in somemalignancies such as prostate cancer the clear association of loss ofp53 function with metastasis suggests the existence of additionalp53-mediated anti-metastatic activities. These results show anadditional role for p53 as an antimetastatic gene as an inducer ofmRTVP-1, which in turn can generate an antitumor immune response that ismanifest locally as well as systemically. One previously publishedreport indicated that p53 could induce the CX3C chemokine, fractalkine,raising the possibility that p53 effector genes are also involved inimmune cell stimulation²⁹. It is of interest that RTVP-1 and fractalkineshare some structural characteristics as predicted by the amino acidsequence. Both proteins have a consensus signal peptide and a putativetransmembrane domain region. As fractalkine has been shown to be presentin a soluble form^(30,31) it appears that this chemokine and potentiallyRTVP-1 exist as both membrane bound and soluble forms, and are thereforecapable of paracrine activities. In the case of fractalkine, paracrinefunctions appear to be limited to chemotactic and pro-adhesionactivities^(30,31) However, the paracrine functions of mRTVP-1 appear togenerate widespread immunostimulatory activities in vivo that includethe induction of F4/80-positive macrophage and CD8+ T cell infiltrateswithin the primary tumor; the induction of splenocyte derived NKactivities; and the generation of increased serum IL-12 levels.

[0079] The uptake of increased numbers of apoptotic bodies by antigenpresenting cells could lead to the activation of specific immune cellsincluding macrophages that were shown to be increased within primarytumors following AdmRTVP-1 treatment. Activated macrophages orpotentially activated dendritic cells could secrete IL-12 (shown to beincreased in serum) and lead to increased systemic NK activities.Alternatively, membrane bound or soluble mRTVP-1 could directly activateimmune cell activities. Since RTVP-1 expression has been associated withdifferentiated macrophages,²⁵ a role for RTVP-1 as a cytokine-likemolecule is likely.

[0080] These data clearly demonstrate that p53 can induce expression ofRTVP-1 in prostate cancer cells in vitro and that overexpression ofmRTVP-1 has widespread immunostimulatory activities in vivo. Further,stress-related p53 induction may induce RTVP-1 levels sufficiently toinduce immune cell activities that counteract the growth and progressionof human prostate cancer.

[0081] These studies are relevant for prostate cancer as the developmentof effective treatments for prostate cancer are frustrated by thenatural history of the disease. The biological and clinical potential ofmost individual cancers is uncertain and in many cases the disease willnot progress to clinical significance. However, experimental andclinical studies indicate that prostate cancer can and may metastasizeearly in the course of the disease from relatively small foci^(32,33).Although localized prostate cancer is potentially curable with radicalprostatectomy or irradiation therapy, there are no curative therapiesfor metastatic prostate cancer. As shown by these results, expression ofmRTVP-1 in prostate cancer through adenoviral vector transfer can inducelocal cytotoxicity through direct and indirect apoptosis whilesimultaneously initiating a local and systemic anti-tumor immuneresponse. Thus, RTVP-based therapy may be useful as a new weapon againstboth prostate cancer and metastatic disease.

Example 6 Mechanism of Action of RTVP-1

[0082] To further explore the underlying mechanisms responsible formRTVP-1 mediated anti-neoplastic activity, additional experiments wereconducted. All experiments were performed as indicated or according toprocedures which are well known to those of ordinary skill in the art.As shown in FIG. 7, p53 has an important role in the progression ofprostate cancer (FIG. 7a), both for a bystander effect (FIG. 7b), andfor regulation of genes in prostate cancer (FIG. 7c). Promoterconstructs of both mutant and wild-type p53 demonstrate that regulationof the mRTVP-1 promoter require p53 binding (FIG. 8). Further, inductionof mRTVP-1 was shown in both RM9 and TSU-Pr-1 cells by Adp53 by gammairradiation (FIG. 9). RTVP-1 expression was evaluated for both mouse andhuman tissues (FIG. 10). Induction of apoptosis was shown in PC-3 cells,LNCaP cells, TSU-Pr1 cells and H1299 cells (FIG. 11), as well asactivation of mitochondrial pathways by mRTVP-1 (FIG. 12), and caspaseactivation (FIG. 13).

[0083] Experiments were also performed demonstrating a role for RTVP-1in apoptosis of prostate cancer. A schematic is shown in FIG. 14, andthe induction of apoptosis in 178-2BMA cells by AdmRTVP-1 in FIG. 15.Mice with orthotopic 178-2BMA tumor tissue treated with AdmRTVP-1 showedincreased survival (FIG. 16), as well as necrosis of the tumor tissues(FIG. 17). Immune cell infiltrates were seen (FIG. 18a) and an immuneresponse for IL-12, NK activity and CTL activity (FIG. 18b). Cytokineinduction was shown for both mouse and human prostate cancer in vitro(FIG. 19), and mRTVP-1 was shown to activate the JNK signal transductionpathway (FIG. 20). Potential mechanisms of mRTVP-1-mediated suppressionof metastasis are shown in FIG. 21. Demethylation of 148-IPA cells withincreasing amounts of 5aza-deoxyC was shown to induce RTVP-1 (FIG. 22).

[0084] Other embodiments and uses of the invention will be apparent tothose skilled in the art from consideration of the specification andpractice of the invention disclosed herein. All references cited herein,including all publications, U.S. and foreign patents and patentapplications including U.S. provisional application No. 60/209,989entitled “P53 Regulated Gene Encoding p44-3 and p44-3 Protein,” filedJun. 8, 2000, are specifically and entirely hereby incorporated hereinby reference. It is intended that the specification and examples beconsidered exemplary only, with the true scope and spirit of theinvention indicated by the following claims. TABLE 1 Homology Comparisonto Mouse RTVP-1 (p144-3 clone) Percent Match H. sapiens mRNA for RTVPprotein 53.7 Human glioma pathogenesis-related protein 60.5 H. sapiensmRNA for cysteine-rich secretory protein 49.6 H. sapiens mRNA forcysteine-rich secretory protein 49.7 H. sapiens mRNA for cysteine-richsecretory protein 47.5 Acidic epididymal glycoprotein gene 49.3 HumanmRNA for acidic epididymal glycoprotein 49.3 Galago crassicaudatusencoding von Willebrand factor 35.2 Rat alpha2 urinary globulin gene23.5 Human P450c21B gene coding for steroid 34.6 Human testes-specificprotein (Tpx-1) 47.1 Mouse surfeit locus surfeit 3 gene 35.3

[0085] TABLE 2 RTVP-1 mRNA expression in human prostate specimens by insitu hybridization RTVP-1 P53 staining^(‡) in RTVP- mRNA 1 positivespecimens % score^(†) % (n) (n) Normal glandular cells +  0 (0/5) ++ 40(2/5) +++ 60 (3/5) Primary cancer cells + 12 (2/16) 50 (1/2) ++  69(11/16)  36 (4/11) +++ 19 (3/16)  0 (0/3) Lymph node metastatic cancercells* + 60 (3/5) 67 (2/3) ++ 40 (2/5)  0 (0/2) +++  0 (0/5)

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1. An isolated nucleic acid comprising all or a portion of a non-humansequence that encodes an RTVP polypeptide wherein said polypeptide hasanti-neoplastic activity.
 2. The nucleic acid of claim 1 which comprisesSEQ ID NO:
 1. 3. The nucleic acid of claim 1 where the sequence isselected from the group consisting of a complete non-human sequence ofRTVP; a portion of the sequence of a non-human RTVP wherein said portionencodes all or an active fragment of a RTVP polypeptide; a nucleic acidencoding at least ten contiguous amino acid residues encoded in anon-human RTVP; and any sequence complementary thereto.
 4. The nucleicacid of claim 1 which is composed of DNA, RNA or PNA.
 5. The nucleicacid of claim 1 wherein the non-human sequence is mammalian.
 6. Thenucleic acid of claim 5 wherein the mammal is a mouse.
 7. The nucleicacid of claim 1 wherein the characteristic portion is selected from thegroup consisting of an open reading frame of the encoded polypeptide, aconserved domain of the nucleic acid, a conserved domain of the encodedprotein, an intron, an exon and combinations thereof.
 8. A vectorcomprising the nucleic acid of claim
 1. 9. A recombinant cell comprisingthe vector of claim
 8. 10. An isolated nucleic acid which encodes atleast an active portion of a nonhuman RTVP protein.
 11. The nucleic acidof claim 10 wherein the active portion is functionally or antigenicallyactive.
 12. An isolated nucleic acid which encodes only a portion of thehuman RTVP protein.
 13. The nucleic acid of claim 12 wherein the portionis selected from the group consisting of functionally active portions,antigenically active portions and combinations thereof.
 14. The nucleicacid of claim 1 wherein the polypeptide has a homology to a mammaliantestes-specific protein, a plant pathogenesis-related protein, a vespidvenom allergan, or a combination thereof.
 15. The nucleic acid of claim14 wherein the homology is selected from the group consisting of greaterthan 35%, greater than 45%, greater than 60% and greater than 70% aminoacid similarity.
 16. The nucleic acid of claim 10 wherein the non-humanRTVP protein encoded has anti-neoplastic activity.
 17. The nucleic acidof claim 16 wherein the anti-neoplastic activity comprises modulation ofa cytokine activity.
 18. The nucleic acid of claim 17 wherein thecytokine activity is selected from the group consisting of activity ofIFN-alpha, -beta and -gamma, TNF-alpha, IL-1β, IL-2, IL-4, IL-6, IL-10,IL-12, Fas, and any receptor thereto, and any combinations thereof. 19.The nucleic acid of claim 16 wherein the anti-neoplastic activitycomprises induction of apoptosis.
 20. The nucleic acid of claim 16wherein the anti-neoplastic activity comprises antiangiogenic activity.21. An isolated polypeptide comprising an amino acid sequence of anon-human RTVP polypeptide wherein said polypeptide has anti-neoplasticactivity.
 22. The polypeptide of claim 21 which comprises SEQ ID NO. 2,or an active portion thereof.
 23. The polypeptide of claim 21 whereinthe amino acid sequence is selected from the group consisting of anexonic region of the polypeptide, a conserved domain of the polypeptide,a functional domain of the polypeptide, an antigenic domain of thepolypeptide, a p53 binding site of the polypeptide, and combinationsthereof.
 24. The polypeptide of claim 22 wherein the active portion is afunctionally or antigenically active portion.
 25. The polypeptide ofclaim 21 which has a homology to a mammalian testes-specific protein, aplant pathogenesis-related protein, a vespid venom allergan, or acombination thereof.
 26. The polypeptide of claim 25 wherein thehomology is selected from the group consisting of greater than 35%,greater than 45%, greater than 60%, and greater than 75% amino acidsimilarity.
 27. The polypeptide of claim 21 wherein the anti-neoplasticactivity is selected from the group consisting of modulation of animmune response, induction of apoptosis, anti-angiogenic activity andcombinations thereof.
 28. A composition comprising the polypeptide ofclaim
 21. 29. The composition of claim 28 further comprising apharmaceutically acceptable carrier selected from the group consistingof water, oils, alcohols, salts, fatty acids, saccharides,polysaccharides and combinations thereof.
 30. A diagnostic kitcomprising the polypeptide of claim 21 for the detection of neoplasticdisease.
 31. The kit of claim 30 wherein the neoplastic disease isprostate cancer or metastatic disease.
 32. An antibody which is reactiveagainst an amino acid sequence of an RTVP polypeptide.
 33. The antibodyof claim 32 which is a monoclonal antibody.
 34. A hybridoma whichproduces the monoclonal antibody of claim
 33. 35. The antibody of claim32 which is reactive against one or more of the group consisting of anexonic region of the polypeptide, a conserved domain of the polypeptide,a functional domain of the polypeptide, an antigenic domain of thepolypeptide, a p53 binding site of the polypeptide, and combinationsthereof.
 36. A vaccine comprising at least a portion of the polypeptideof claim
 21. 37. A diagnostic kit comprising the antibody of claim 32for the detection of neoplastic disease.
 38. The kit of claim 37 whereinthe neoplastic disease is prostate cancer or metastatic disease.
 39. Amethod for treating a patient comprising administering to the patient atherapeutically effective amount of a composition comprising at least anactive portion of the polypeptide of claim
 21. 40. The method of claim39 wherein the polypeptide has anti-neoplastic activity.
 41. The methodof claim 39 wherein the anti-neoplastic activity is selected from thegroup consisting of modulation of an immune response, induction ofapoptosis, anti-angiogenic activity and combinations thereof.
 42. Themethod of claim 41 wherein the immune response comprises modulation of acytokine selected from the group consisting of IFN-alpha, -beta and-gamma, TNF-alpha, IL-1β, IL-2, IL-4, IL-6, IL-10, IL-12, Fas, and anyreceptor thereto, and any combination thereof.
 43. The method of claim39 wherein the patient is a mammal.
 44. An isolated RTVP receptorprotein or portion thereof which binds to the polypeptide of claim 21.